1. Field of the Invention
This invention relates to immunoassays and to such assays in which a fluorescent tag capable of emitting fluorescent radiation when excited by more energetic exciting radiation is incorporated into a constituent of an antigen-antibody or similar complex. More particularly, this invention relates to optical apparatus used for performing the assay and still more particularly to improved optical apparatus which focuses the excitation radiation onto a substrate coated with the fluorescent material limited substantially to the region at which evanescent wave coupling occurs.
2. Description of the Prior Art
Immunoassay, in which aliquots of sample and one or more reagents are variously reacted to form antigen-antibody or similar complexes which may then be observed in order to assay the sample for the presence and titer of a predetermined moiety of the complex, are well known. Typical of such assays are those wherein a specific antibody is used to measure the quantity of the antigen for which it is specific, or vice versa. However, the technique has been extended to quantitate haptens including hormones, alkaloids, steroids, and the like as well as antigens, and antibody fragments (i.e., Fab) as well as complete antibodies, and it is in this broader sense that the present invention should be understood.
As is well known, sensitive immunoassays employ tracer techniques Wherein a tagged constituent of the complex is incorporated into the reagent, the non-complexed tagged reagent being separated from the complexed reagent, and the complex (or non-complexed reagent) then quantitated by observing the tag. Both radioisotopes and fluorescent markers have been used to tag constituents of immunoassay reagents, the tag being respectively observed by a gamma, ray counter or a fluorometer. The present invention is, however, directed only to those assays which rely on fluorescence.
The separation of the non-complexed tagged moiety from the complexed is commonly accomplished by immobilizing a predetermined one of the components of the complex to a solid phase (such as the inside wall of a test tube, glass or polymeric beads, or the like) in such a way as not to hinder the component's reactivity in forming the complex. As an example, an antibody such as immunoglobulin G (IgG) may be bound by its carboxyl terminations to a solid phase, such as glass, by a silyl compound such as 3-aminopropyltrimethoxysilane, thereby leaving free the antigen reactive amino terminations of the antibody. Any complex formed incorporating the immobilized component may then be physically separated from the non-reacted complement remaining in solution, as by aspirating or decanting the fluid from a tube or eluting the fluid through a particulate bed.
In competition immunoassay, the reagent consists of a known quantity of tagged complement (such as antigen) to the immobilized component of the complex (in this instance, antibody). The reagent is mixed with a fixed quantity of the sample containing the untagged complement to be quantitated. Both tagged and untagged complement attach to the immobilized component of the complex in proportion to their relative concentrations. After incubation for a set time, the fluid sample and reagent are separated. The complex immobilized to the solid phase is then illuminated with radiation of a wavelength chosen to excite fluorescence of the tag, and the fluorescence is measured. The intensity of the fluorescence of the immobilized complex is inversely proportional to the concentration of the untagged complement being assayed.
Alternatively, an assay may be made by immobilizing a quantity of an analog of the moiety to be quantitated (i.e., a substance which is immunologically similarly reactive) and reacting the sample with a known quantity of tagged complement. The tagged complement complexes with both the unknown quantity of the moiety in the sample and the immobilized analog. Again, the intensity of fluorescence of the immobilized complex is inversely proportional to the concentration of the (free) moiety being quantitated.
So-called "sandwich" immunoassays may be performed for multivalent complements to the immobilized component, the attached complement being then further reacted with a tagged analog of the immobilized component. Thus, bivalent antigen may be bound to an immobilized antibody and then reacted with a fluorescent tagged antibody, forming an antibody-antigen-tagged antibody sandwich that may then be separated from the unreacted tagged antibody. The intensity of the fluorescence of the thus formed immobilized complex is directly proportional to the concentration of the species being quantitated.
In any of the assays, accuracy in quantitation is determined in part by the technique of the laboratory personnel performing the assay. Thus, precision fluorescence immunoassay requires fluorometric measurement of a predetermined volume of the sample to which a predetermined quantity of reagent has been added at a known dilution. To insure that the necessary volume measurements are not the accuracy limiting step of the assay requires that the assay be performed by skilled personnel and often with precision apparatus, or alternatively, precisely constructed and preloaded disposable reagent kits (to insure the titer of reagent) together with an accurately timed diffusion process (to insure the size of the sample volume assayed.
It will be appreciated that the use of skilled personnel, precision apparatus, and accurate manipulative or timing requirements impact both the cost and wide-scale availability of an assay.
It is well known that optical systems employing the principles of attenuated total internal reflection (ATR) spectroscopy, are useful in chemical and biochemical analysis or assay including immunoarrays. For example, U.S. Pat. No. 4,133,639 discloses a system based on absorption of the evanescent wave by the analyte; U.S. Pat. Nos. 4,321,057 and 4,399,099 both disclose systems that detect changes in the radiation transmitted through the fiber; and U.S. Pat. No. 4,447,546 describes a fluorescence immunoassay system.
In apparatus as described in the aforementioned U.S. Pat. No. 4,447,546, an optical fiber is supported within a capillary tube in approximately concentric alignment therewith. A fluid sample is introduced into the interspace between the fiber and the tube and is drawn into and supported in the interspace by capillary action. To maximize sensitivity and efficiency of such an immunoassay apparatus, it is important that the fiber remain spaced from the internal walls, of the capillary tube. If the fiber contacts the capillary wall, capillary action may be adversely affected. and total internal reflection will not be achieved since radiation will leak out of the fiber at the point of contact between the fiber and the capillary wall with attendant loss of sensitivity.
It is important that the end of the fiber into which optical radiation and from which fluorescent radiation are transmitted be supported in a fixed axial position with respect to an optical system for transmitting optical radiation in and out of the fiber. In the event that end of the fiber does not lie at a fixed position with respect to that optical system, the amount and orientation of transmitted radiation entering the fiber may vary, adversely affecting the accuracy and sensitivity of the apparatus.
Several techniques have been developed in known immunoassay apparatus for properly positioning an optical fiber within a capillary tube. The oldest technique involves supporting the proximal end (i.e. the end into which radiation :s initially launched) of the optical fiber using a conventional Fiber optic connector. Use of these connectors typically involves covering the outer surface of the fiber adjacent its proximal end with a cladding material typically consisting of a transparent high molecular weight polymer. Known cladding materials typically have a refractive index higher than that of the sample, e.g. 1.40 to 1.45, with the result that the numerical aperture of the fiber is reduced to a level at which acceptable sensitivity levels cannot readily be achieved with the apparatus.
Another technique, described in U.S. Pat. No. 4,671,938, involves supporting the fiber in cantilever fashion at its distal end, i.e. the end opposite the end where optical radiation is transmitted into the fiber. The proximal end of an optical fiber supported in this fashion is however displaceable both axially and radially, and such displacement will also cause loss of instrument sensitivity. Further, when the fiber is enclosed in a capillary tube so that a liquid sample being assayed can be introduced into the interspace between the fiber and the capillary tube, the end of the tube surrounding the proximal end of the fiber has not heretofore been readily sealable to prevent leakage of that sample. The toroidal fluid meniscus formed at the end of the tube can serve to prevent fluid flow out of that end of the capillary tube but will, of course, tend to break down when subjected to shock. vibration. high pressure and the like. If the sample being assayed is highly toxic or infectious, such a casual barrier is unacceptable.
In yet another technique for supporting the fiber, the fiber and surrounding capillary tube are disposed in mounting apparatus for attachment to an optical assembly, for transmitting excitation radiation into the proximal end of the fiber and receiving fluorescent radiation emitted from the proximal end of the fiber. Included in the apparatus is a mounting assembly for centering the fiber within the capillary), tube and for biasing the fiber in a first direction against an annular seat. The latter is designed to support one end of the fiber so that none of the radiation introduced into the fiber is intercepted by the seat.
In such total internal reflection systems, the evanescent zone around the fiber increases in depth and the sensitivity of the system also increases as the numerical aperture of the fiber increases. Also the intensity of the fluorescent signal tunnelling back into the fiber is proportional to a very high power of the numerical aperture (as defined in part by the refractive index of the sample in which fluorescence is excited). Thus. it is preferred that the numerical aperture of the system be maximized, particularly by providing the input radiation at as high a flux as possible over a maximum solid acceptance angle. Such maximization has heretofore been limited by the first of the above-described techniques used to clamp and support the fiber, particularly where the diameter of the fiber employed is very small, e.g. 300-400 microns. To obtain very high numerical apertures using a separate mounting assembly, the art has heretofore typically employed highly corrected lenses with shallow depth of field. Such lenses are expensive, and are difficult to manufacture and to maintain in alignment.